PROJECT SUMMARY Chromatin structure is highly dynamic and can be modulated through a number of different mechanisms, including nucleosome remodeling, histone post-translational modifications and, relevant to this proposal, the nucleosomal incorporation of histone variants. This proposal focuses on the macroH2A histone variants, which are primarily associated with condensed chromatin and inactive genes, and act as tumor suppressors across a spectrum of cancers (Vardabasso et al., 2014). We showed that macroH2A variants inhibit tumor growth and metastatic potential of melanoma cells (Kapoor et al., 2010). Here we will use a unique macroH2A double knockout (dKO) mouse model (Pehrson et al., 2014) to study cancer initiation and progression in the absence of macroH2A variants in vivo. We previously used these mice to demonstrate that macroH2A isoforms present a barrier to the reprogramming process of somatic cells towards a pluripotent state (Gaspar-Maia et al., 2013). However, macroH2A-deficient animals have yet to be crossed with any genetically engineered mouse models of cancer. Owing to the importance of macroH2A in suppressing melanoma progression and our unpublished studies showing a defect in mammary gland differentiation coupled to increased stem cell activity, we hereby propose to model melanoma and breast cancer in the context of a living organism. In Aim 1, we will investigate the role of macroH2A in suppressing melanoma pathogenesis by crossing macroH2A dKO mice to an established melanoma mouse model (Tyr-Cre/BRAFV600E/Pten-flox) (Dankort et al., 2009). We will investigate whether loss of macroH2A isoforms promotes tumor growth and/or metastasis in the induced tumors. These analyses will be coupled to xenograft models, as well as genetically defined melanocytes and melanoma cell lines in vitro for probing the underlying molecular mechanisms of macroH2A tumor suppression via genomic approaches. In Aim 2, we will challenge the macroH2A-deficient mice by crossing them to an established mammary tumor model (MMTV- NeuY1227D). Given the mammary gland phenotype we identified in the dKO mice, as well as the loss of macroH2A isoforms in human breast cancer specimens, we hypothesize that in the presence of mammary driver mutations, macroH2A dKO mice will develop more proliferative and invasive cancers. These studies will also be coupled to genomics approaches. In both aims, we will determine which macroH2A isoforms, domains and modified residues allow inhibition of tumor development by reconstituting macroH2A in mouse and human cells devoid of this histone variant. This unique mouse colony provides us with exciting opportunities for studying macroH2A histone variant regulation in development and cancer, allowing for a deeper understanding of human disease.